PERSPECTIVES BIOCHEMISTRY Fifty years of research have led to a detailed understanding of the mechanisms of enzymatic catalysis. How Enzymes Work Dagmar Ringe and Gregory A. Petsko azing at the three-dimeneven if almost every other facsional structures of enzytor were eliminated by mutating mes that regularly grace the enzyme, the protein would the covers of scientific publicastill be a respectable catalyst. tions, it is hard to imagine that Second, Koshland was right: there are still people alive who The active-site residues usually remember when many biochemadjust to permit the binding Asp52 ists thought that enzymes had no of the specific substrate. Inordered structure. But that was the duced-fit changes involving case until James Sumner crystalthe movement of entire protein lized urease in 1926 (1)—a develdomains by several nanometers opment so revolutionary that he have been observed (6). Third, was taken into custody as a danthe protein structure can create gerous lunatic when he tried to specialized microenvironments explain what he had done to a that dramatically alter the reac35 Glu famous European scientist. When tivity of key catalytic groups, in biochemists realized that enzymes some cases by shielding the had persistent structure and that catalytic site from contact with Elucidating the active site. In the crystal structure of a lysozyme mutant bound to destruction of that structure could a synthetic sugar substrate, the sugar ring in the active site is distorted, and the scis- bulk solvent. Fourth, enzymes abolish enzyme activity, they rap- sile bond is close to the acid-base residues Asp52 (left) and Glu35 (lower right; can distort the substrate, causidly adopted the view that enzymes mutated to Gln in this structure) (5). All these features were deduced by Phillips and ing it to adopt a high-energy were rigid scaffolds whose speci- co-workers more than 40 years ago (4). Unexpectedly, the structure also shows that conformation with increased ficity and catalytic power came lysozyme can form a covalent intermediate with its substrates (5). reactivity (7). Finally, enzymes from the inflexible fit of the right provide extra stabilizing intersubstrate onto the preformed enzyme surface, and biophysical experiments. The induced fit actions for the transition state (or unstable interthe way a key fits a lock. Fifty years ago, Daniel hypothesis was still controversial, and most mediates) in the reaction mechanism. Specific Koshland challenged this view, proposing that models of enzyme function postulated a fairly stabilization of the transition state, particularly the enzyme surface was flexible and that only rigid catalyst. Proximity—the holding of sub- electrostatically, is thought to be so important the specific substrate would induce the proper strate molecules and catalytic groups on the that an entire industry—the development of interactions that led to catalysis (2). enzyme in close approximation and in orien- catalytic antibodies—has been based on this Studies of enzyme mechanisms were driven tations favoring the appropriate bond-break- single principle (8–10). by a wish to understand the ability of enzymes ing and bond-making steps—was generally Most, if not all, enzymes derive the bulk of to accelerate the rate of a chemical reaction by held to have an important role in catalysis, but their catalytic power from varying combinastaggering amounts—up to 1020 times the rate other details were murky. tions of these simple factors. Confirming eviof the uncatalyzed reaction in water (3)—while The fog lifted, brilliantly, over the course dence has come from a wide range of elegant displaying a specificity so tight that some of a single weekend, when Phillips took the experiments, notably site-directed mutagenesis, enzymes can discriminate between sulfate and atomic model of his newly determined which allows specific groups on the enzyme phosphate. As we celebrate not only the lysozyme structure, built into its active site a to be changed or removed (11–13), and high50th anniversary of Koshland’s “induced fit” model of the oligosaccharide substrate, and resolution x-ray crystallography, especially of hypothesis but also ~50 years of high-resolu- deduced a set of structural factors that he enzyme-substrate and enzyme-intermediate tion protein structure determination by x-ray believed could explain the ability of this complexes (14). crystallography, it is instructive to look back on enzyme to digest the peptidoglycan cell walls What was missing in this picture? Three the history of attempts to explain enzymatic of many bacteria. Forty years of follow-up relatively recent discoveries stand out. One is catalysis and to summarize what we understand experiments proved his inspired reasoning the contribution of quantum mechanical tuntoday about how these remarkable macromole- correct in almost every detail, although a neling to the rates of enzyme-catalyzed reaccules function. recent study provides a new wrinkle (see the tions whose mechanisms involve the transfer Before the first crystal structure of an figure) (5). Moreover, the factors he enumer- of hydrogen ions (15). Another is the precise enzyme was determined, that of lysozyme by ated turned out to be applicable to almost all matching of the pKa’s (a logarithmic measure David Phillips and his team in 1965 (4), spec- other enzymes. of the proton affinity of a weak acid) of the ulations about how enzymes worked were What are the lessons from lysozyme? First, donor and acceptor atoms in hydrogen bonds based on deductions from indirect biochemical proximity and orientation are critical. Much of that stabilize the transition state. Such matchwhat an enzyme does is to bring the reacting ing can lead to short, symmetrical hydrogen species together in a geometry that favors reac- bonds of greater-than-normal strength (16, 17). Department of Biochemistry, Brandeis University, Waltham, MA 02454, USA. E-mail: [email protected] tion. This is so important that in some cases, But perhaps the most active area of current 1428 13 JUNE 2008 VOL 320 SCIENCE Published by AAAS www.sciencemag.org Downloaded from www.sciencemag.org on September 4, 2008 G research is the possible role of protein dynamics in aiding the reacting species in crossing the transition-state barrier to the reaction. As originally formulated, the structure of the enzyme was proposed to favor atomic vibrations along the reaction coordinate while disfavoring those that would not lead to productive bond-making or bondbreaking steps (18). Recent evidence from different enzyme systems suggests that this factor may indeed contribute to catalytic efficiency (19, 20). Given that we now have a good understanding of the principles underlying enzyme catalytic proficiency and specificity, it seems appropriate to ask where the field is likely to go next. Practical applications, such as the creation of enzymes catalyzing novel reactions, are under way. Further investigations into the role of protein dynamics in enzymatic catalysis are still needed. But we believe that a crucial next step will be to go beyond the milieu of dilute aqueous solution and individual purified enzymes that has defined enzymology for the past 100 years. Most enzymes function in the interior of the cell, where the substrate concentration is typically very low and the protein concentration may exceed 100 mM. How do enzymes function in a crowded medium of low water activity, where there may be no such thing as a freely diffusing, isolated protein molecule? In vivo enzymology is the logical next step along the road that Phillips, Koshland, and their predecessors and successors have traveled so brilliantly so far. References and Notes 1. J. B. Sumner, J. Biol. Chem. 69, 435 (1926). 2. D. E. Koshland Jr., Nature 432, 447 (2004). 3. C. Lad, N. H. Williams, R. V. Wolfenden, Proc. Natl. Acad. Sci. U.S.A. 100, 5607 (2003). 4. C. C. Blake et al., Proc. R. Soc. London B 167, 378 (1967). 5. D. J. Vocadlo, G. J. Davies, R. Laine, S. G. Withers, Nature 412, 835 (2001). BIOCHEMISTRY 6. T. A. Steitz, R. Harrison, I. T. Weber, M. Leahy, Ciba Found. Symp. 93, 25 (1983). 7. D. L. Pompliano, A. Peyman, J. R. Knowles, Biochemistry 29, 3186 (1990). 8. S. D. Lahiri, G. Zhang, D. Dunaway-Mariano, K. N. Allen, Science 299, 2067 (2003). 9. A. Warshel et al., Chem. Rev. 106, 3210 (2006). 10. R. A. Lerner, C. F. Barbas III, K. D. Janda, Harvey Lect. 92, 1 (1996–1997). 11. J. R. Knowles, Nature 350, 121 (1991). 12. T. C. Bruice, S. J. Benkovic, Biochemistry 39, 6267 (2000); 13. D. A. Kraut, K. S. Carroll, D. Herschlag, Annu. Rev. Biochem. 72, 517 (2003). 14. I. Schlichting et al., Science 287, 1615 (2000). 15. Z. D. Nagel, J. P. Klinman, Chem. Rev. 106, 3095 (2006). 16. W. W. Cleland, P. A. Frey, J. A. Gerlt, J. Biol. Chem. 273, 25529 (1998). 17. D. A. Kraut et al., PLoS Biol. 4, e99, (2006). 18. T. Alber et al., CIBA Found. Symp. 93, 4 (1982). 19. S. Hammes-Schiffer, S. J. Benkovic, Annu. Rev. Biochem. 75, 519 (2006). 20. K. A. Henzler-Wildman et al., Nature 450, 838 (2008). 21. We dedicate this paper to the memory of our good friend and long-time collaborator Jeremy R. Knowles. 10.1126/science.1159747 New results provide support for the hypothesis that interactions between proteins involve selection from an ensemble of different conformations. How Do Proteins Interact? David D. Boehr and Peter E. Wright nteractions between proteins are central to biology and are becoming increasingly important targets for drug design. Upon forming complexes, protein conformations usually change substantially compared to the unbound protein. Two main hypotheses have been advanced to explain these changes (see the figure). According to the “induced fit” hypothesis, the initial interaction between a protein and a binding partner induces a conformational change in the protein through a stepwise process (1). In the “conformational selection” model, it is assumed that, prior to the binding interaction, the unliganded protein exists as an ensemble of conformations in dynamic equilibrium. The binding partner interacts preferentially with a weakly populated, higher-energy conformation-causing the equilibrium to shift in favor of the selected conformation. This conformation then becomes the major conformation in the complex (2). Although biochemistry textbooks have championed the induced fit mechanism for more than 50 years, there is now growing support for the additional bind- I Department of Molecular Biology and Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037, USA. E-mail: [email protected]; wright@ scripps.edu ing mechanism, including the seminal work by Lange, Lakomek, and co-workers on page 1471 of this issue (3). A major stumbling block for the conformational selection hypothesis has been the inability to characterize the structures of the predicted multiple conformations (or conformational substates) of a protein. The structural models resulting from x-ray crystallography tend to identify only a single dominant conformation, although different crystal forms of the same protein can provide insights into the range of conformations accessible to the protein (4). Help comes from nuclear magnetic resonance (NMR), a powerful method for characterizing protein dynamics and the protein conformational ensemble at the atomic level. Various NMR observables (5, 6) give structural information about lowly populated, higher-energy conformations that are invisible to other techniques. In a previous report, Vendruscolo and coworkers (7) combined data from NMR relaxation experiments with molecular dynamics simulations to characterize a structural ensemble of the protein ubiquitin. However, the experimental data only covered nanosecond time-scale dynamics and thus failed to capture the slower time scales that are important for molecular recognition. www.sciencemag.org SCIENCE VOL 320 Published by AAAS Lange et al. have now extended the methodology to slower time scales by using residual dipolar couplings (RDCs) (3), which serve as restraints for structural determination by NMR and also provide dynamic information over a wide range of time scales (8). By analyzing RDCs measured for a large range of solution conditions, Lange et al. construct a structural ensemble for ubiquitin that describes its dynamic behavior up to the microsecond time scale. The most striking feature of the ensemble is the presence of conformations that are nearly identical to the 46 known bound forms of ubiquitin observed in x-ray crystal structures. The results provide very strong evidence that complex formation by ubiquitin involves conformational selection processes. Gsponer et al. recently reported a similar result for calmodulin. Using the methodology of Vendruscolo and co-workers, they showed that the nanosecond ensemble for apocalmodulin contains conformations similar to calmodulin bound to myosin light chain kinase (9). The structural ensemble reported by Lange et al. is consistent with the energy landscape theory of protein folding and function (2, 10, 11). This theory posits that there are multiple protein conformations in dynamic equilib- 13 JUNE 2008 Downloaded from www.sciencemag.org on September 4, 2008 PERSPECTIVES 1429